Optimization of Purification Protocols Based on the Step-by-Step Monitoring of the Protein Aggregates in Soluble Fractions
Soluble protein fractions are often considered containing exclusively monodispersed and correctly folded molecules. This is not the case, being soluble aggregates of different complexity widely represented in such fractions. The use of fusing target protein domains to highly soluble carriers may strongly contribute to soluble aggregate accumulation. Therefore, reliable analytical methods must be used to evaluate the biophysical characteristics of soluble proteins. On the other hand, conventional methodologies are often technically demanding and time consuming. In this method paper, a protocol is presented that enables the rapid evaluation of the protein monodispersity from the initial step aimed at screening several conditions in parallel to the setup of the complete protocol for large-scale purification. The analysis is performed by means of simple lab equipment and starting from small sample volumes.
Key wordsAggregation index Light scattering Magnetic beads Size-exclusion chromatography Soluble aggregates
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